A Metal Bridge between Two Enzyme Families

Duewel, Henry S.; Woodard, Ronald W.

doi:10.1074/jbc.m000133200

Cited by 50 publications

(26 citation statements)

References 63 publications

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“…As discussed by Duewel et al (15,16), this color is probably due to the presence of substoichiometric amounts of bound iron in the enzyme produced in E. coli. For these studies, we have opted to collect diffraction data from crystals that were either completely depleted of any metal or in which the iron had been replaced with Cd 2ϩ , the ion that confers the highest activity in solution (15). Incubation of A. aeolicus KDO8PS crystals in a cryoprotectant liquor containing 100 M CdCl 2 , with several daily changes of this solution, bleaches their color completely within 2 days.…”

Section: Methodsmentioning

confidence: 93%

“…The Cd 2ϩ form (Table I, Cd 2ϩ column) was intentionally pursued because Cd 2ϩ is the most effective activator of KDO8PS (15). In the active site of the enzyme, the Cd 2ϩ ion displays a distorted octahedral coordination: the thiolate of Cys-11 and the ⑀-nitrogen of His-185 provide two axial ligands, whereas the carboxylate moieties of Glu-222 and Asp-233 and a water molecule provide the equatorial coordination (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…has been previously reported that E4P, the substrate of DAH7PS, does not serve as a substrate for A. aeolicus KDO8PS (15,16). Furthermore, in the E. coli enzyme, E4P acts as a competitive inhibitor with respect to A5P.…”

Section: Structure Of the Enzyme In Complex With Pep Plus E4p-itmentioning

confidence: 99%

“…The most extensively studied member of the KDO8PS family, the E. coli enzyme, does not require a metal. In contrast, KDO8PS from the hyperthermophile Aquifex aeolicus requires a divalent cation for activity, with the level of activation varying over a 10-fold range in the order Cd (15). To understand the basis for the different metal requirement between these two enzymes, we have determined the crystal structure of A. aeolicus KDO8PS in the presence of various combinations of substrates and metal.…”

Section: ؉mentioning

confidence: 99%

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Substrate and Metal Complexes of 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase from Aquifex aeolicus at 1.9-Å Resolution

Duewel

Radaev

Wang

et al. 2001

Journal of Biological Chemistry

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157

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3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS) from the hyperthermophilic bacteriumAquifex aeolicus differs from its Escherichia coli counterpart in the requirement of a divalent metal for activity (Duewel, H. S., and Woodard, R. W. (2000) J. Biol. Chem. 275, 22824 -22831). Here we report the crystal structure of the A. aeolicus enzyme, which was determined by molecular replacement using E. coli KDO8PS as a model. The structures of the metal-free and Cd 2؉forms of the enzyme were determined in the uncomplexed state and in complex with various combinations of phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and erythrose 4-phosphate (E4P). Like the E. coli enzyme, A. aeolicus KDO8PS is a homotetramer containing four distinct active sites at the interface between subunits. The active site cavity is open in the substratefree enzyme or when either A5P alone or PEP alone binds, and becomes isolated from the aqueous phase when both PEP and A5P (or E4P) bind together. In the presence of metal, the enzyme is asymmetric and appears to alternate catalysis between the active sites located on one face of the tetramer and those located on the other face. In the absence of metal, the asymmetry is lost. Details of the active site that may be important for catalysis are visible at the high resolution achieved in these structures. Most notably, the shape of the PEPbinding pocket forces PEP to assume a distorted geometry at C-2, which might anticipate the conversion from sp 2 to sp 3 hybridization occurring during intermediate formation and which may modulate PEP reactivity toward A5P. Two water molecules are located in van der Waals contact with the si and re sides of C-2 PEP , respectively. Abstraction of a proton from either of these water molecules by a protein group is expected to elicit a nucleophilic attack of the resulting hydroxide ion on the nearby C-2 PEP , thus triggering the beginning of the catalytic cycle.3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS 1 ; phospho-2-dehydro-3-deoxyoctonate aldolase, EC 4.1.2.16) catalyzes the aldol-type condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate ( Fig. 1) (1). This reaction is the first step in the biosynthesis of 3-deoxy-Dmanno-octulosonate, an essential component of the lipopolysaccharide of all Gram-negative bacteria (2). A reaction very similar to KDO8P synthesis is the formation of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) from erythrose 4-phosphate (E4P) and PEP catalyzed by DAH7P synthase (DAH7PS; phospho-2-dehydro-3-deoxyheptonate aldolase, EC 4.1.2.15), which constitutes the first step of the shikimate pathway (3). Earlier studies have established that KDO8PS and DAH7PS share several mechanistic characteristics. For example, in both enzymes, the condensation step of the reaction is stereospecific, involving the addition of the si face of C-3 PEP to the re face of the A5P/E4P carbonyl (4 -7). In particular, KDO8PS and DAH7PS are two of only four known PEP-utilizing e...

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Section: Methodsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Structure Of the Enzyme In Complex With Pep Plus E4p-itmentioning

confidence: 99%

Section: ؉mentioning

confidence: 99%

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Substrate and Metal Complexes of 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase from Aquifex aeolicus at 1.9-Å Resolution

Duewel

Radaev

Wang

et al. 2001

Journal of Biological Chemistry

Self Cite

157

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“…The effect of metal cations on DMS monooxygenase activity in cell-free extracts (CFEs) was assessed. Sequestration of cations from CFE samples was achieved using the EDTA method of Duewel and Woodard (14). Divalent metal cations (e.g., magnesium sulfate, calcium chloride, cupric sulfate, ferrous sulfate, cobaltous chloride, mercuric chloride, manganous sulfate, cadmium chloride, or zinc chloride) were replaced by incubation, at a final concentration of 1 mM, with sequestrated CFE samples for 30 min on ice prior to assessing enzyme activity.…”

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confidence: 99%

Purification and Characterization of Dimethylsulfide Monooxygenase fromHyphomicrobium sulfonivorans

et al. 2011

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Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase from Hyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH 2 -dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.Dimethylsulfide (DMS) is a volatile organosulfur compound, important in the biogeochemical cycling of sulfur and global climate regulation (4, 9). Bacterial metabolism of DMS is an important sink of the compound in nature and is thought to account for degradation of over 80% of the DMS produced in the marine environment. Although bacterial pathways of DMS degradation have been studied previously in Hyphomicrobium spp. and in Thiobacillus spp. (12, 36), they remain poorly characterized, and few enzymes of DMS metabolism have been purified (see reference 32). DMS monooxygenase was first reported from an assay of NADH-dependent oxygen uptake in the presence of DMS by cell extracts of Hyphomicrobium S (12), an activity also demonstrated in cell extracts of other Hyphomicrobium, Thiobacillus, and Arthrobacter isolates (6,7,34), with specific activities around 30 nmol NADH oxidized min Ϫ1 mg protein Ϫ1 . The enzyme has not previously been purified or characterized.The aims of this study were to purify and characterize the DMS monooxygenase enzyme from a member of the genus Hyphomicrobium. Since Hyphomicrobium S is no longer available, studies were undertaken using the type strain of H. sulfonivorans. The strain was originally isolated from garden soil and grows on DMS, as well as the related compounds dimethyl sulfoxide (DMSO) and dimethylsulfone (DMSO 2 ). During growth on DMSO 2 , H. sulfonivorans first reduces DMSO 2 to DMSO by a dimethylsulfone reductase, and subsequently a DMSO reductase converts DMSO to DMS, which is further oxidized to methanethiol and formaldehyde by a DMS mono-

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(−)‐Daucinsäure: Revision der Konfiguration, Synthese und Folgerungen zur Biosynthese

2003

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Synthesen der Dihydropyrane mit D‐xylo‐ (1), D‐ribo‐, L‐arabino‐ und D‐lyxo‐Konfiguration (2) – durch C1‐Homologisierung von D‐Galactose bzw. D‐Mannose, terminale Oxidationen und gezielte β‐Eliminierung – erbrachten den Nachweis, dass der aus Karotten isolierten (−)‐Daucinsäure die D‐lyxo‐, nicht die bislang zugeordnete D‐xylo‐Konfiguration zukommt. Konfigurationsidentität von 2 mit KDO‐8P (3) und vorliegende Untersuchungen zur Biogenese von Chelidonsäure (4) legen für 2 und 4 einen gemeinsamen, auf 3 basierenden Biosyntheseweg nahe.

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A Metal Bridge between Two Enzyme Families

Cited by 50 publications

References 63 publications

Substrate and Metal Complexes of 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase from Aquifex aeolicus at 1.9-Å Resolution

Substrate and Metal Complexes of 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase from Aquifex aeolicus at 1.9-Å Resolution

Purification and Characterization of Dimethylsulfide Monooxygenase fromHyphomicrobium sulfonivorans

(−)‐Daucinsäure: Revision der Konfiguration, Synthese und Folgerungen zur Biosynthese

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